The Confocal Imaging Facility (CIF) of the NIA IRP has been in operation since October 2004. This facility started with one older model (Zeiss LSM 410) confocal system and has successfully integrated a state of the art Zeiss LSM 510 Meta confocal system. In addition, we have updated the Zeiss Axioskop fluorescent microscope to a dual use system: MCID/QImage analysis system and Metamorph/Coolsnap imaging and analysis system. This expansion and upgrading has resulted in extensive collaboration with intramural scientists at different levels of expertise ranging from principal senior investigators through postdoctoral fellows and intramural research trainees. We have trained over 45 investigators to be independent users of the confocal microscope and have held scores of consultations with NIA Lab Chiefs, PIs and researchers to assist with their imaging and image analysis. We have helped users integrate imaging into their experimental systems and in troubleshooting problems. This has resulted in a yearly increase in the use of immunofluorescence and imaging at the IRP, as attested by the widespread use of the CIF by over 60 researchers in FY09, ranging from post-bacs to senior investigators. The following techniques have been used or introduced at the CIF: (a) Use of confocal microscopy for precise sub-cellular localization and co-localization of proteins;(b) Advanced immunological, biochemical and imaging techniques required for the investigation of intracellular and intranuclear protein trafficking;(c) Development of methodology to cause DNA damage to live cell DNA at the sub-micron level by use of the continuous scanning UV laser of the LSM 510 Meta system;(d)Time-lapse, FRAP and ratio-metric analysis of cellular processes in live cells using the 710 confocal microscope;(e) Volumetric (3D) reconstruction of intracellular protein distribution using confocal or deconvolution techniques;(f) Volumetric (3D) reconstruction and surface rendering of human pancreatic islets of Langerhans from optical Z-sections. The importance of these techniques is emphasized in the recent research of the CIF and our collaborators (see Bibliography below, 1-12). We have followed the intracellular trafficking of several proteins, together with the precise sub-cellular localization and high-resolution co-localization of these proteins in multi-component protein complexes. Thus, in collaboration with the lab of Dr. Weeraratna (LI), these methods have yielded very important characterizations of invasive melanoma cells: (a) defining Wnt5a (a protein that increases melanoma metastasis) interaction with syndecan which is via sugar chains, and was not amenable to immunonoprecipitation analysis, but which were proven to bind using immunofluorescence and confocal microscopy (1, 6);(b) Using primarily immunofluorescence and confocal microscopy, we show that phosphorylation modifications of the tight junction protein claudin-1 cause its translocation to the cytoplasm and nucleus and that the sub-cellular localization of claudin-1 may dictate the metastatic capacity of melanoma cells. Our findings suggest that nuclear versus cytoplasmic expression of claudin-1 may become a valuable marker for diagnosis of malignant melanoma (French et al., Int. J. Med. Sci. 6:93-101, 2009);(c) Trafficking of EGFR-GFP showed endocytosis to late endosomes and lysosomes in cells expressing filamin A (an actin-binding protein), but not in cells that do not express filamin A, suggesting that filamin A contributes to activation and sorting of EGFR, an important member of the receptor tyrosine kinase family, that is implicated in oncogenesis(Fiori et al., Endocrinology 150:2551-60, 2009). Another important protein complex was discovered in a different system. In research done in collaboration with the lab of Dr. Biragyn (LI), we used in a T cell system a very nice three-color co-localization technique to provide quantitative evidence for the interaction of CD45 (a phosphatase that regulates Lck), Lck (a Src kinase) and GCR (glucocorticoid receptor) in a cell membrane protein complex that is important for the activation of T cells (Baatar et al., Brain Behav. Immunol. 23:1028-37, 2009). In intranuclear research we have assisted in defining the interactions of RNA binding proteins (2, 7) with our collaborators at the University of Maryland. In another important intranuclear imaging project done in collaboration with the lab of Dr. Ko (LG), we performed a major imaging analysis at high resolution of chromosome telomeres that resulted in publication of a research paper in the journal Nature (3) last year. CIF staff (Sarah Subaran, B.Sc.) contributed to another high-profile research paper published last year in the journal Nature Structural and Molecular Biology (5). This research, performed in collaboration with the lab of Dr. Gorospe (LCMB), involves imaging RNA binding proteins that form cytoplasmic structures called stress granules (8). These structures are defined exclusively by immunofluorescence, underlying the importance of imaging to the mission of the intramural research at the NIA. Currently, the CIF is involved in several projects of the Diabetes Section of the LCI, including the investigation of megalin, a clinically important kidney protein. Megalin (LRP2) is a cell surface receptor present on the membranes of kidney proximal tubule cells and other brush border membranes. It is responsible for the clearance of many polypeptides from blood plasma, including insulin, angiotensin II and leptin. We will use advanced confocal microscopy techniques to investigate the function and trafficking of megalin in live cells, coupled with extensive validition by biochemical methods.